The long term objective of this proposal is to characterize the autoregulatory control mechanisms of bone blood flow with particular emphasis on the relation of these mechanisms to impaired bone blood flow following post-ischemic reperfusion. The investigations completed thus far have clarified the relative importance of alpha-1, alpha-2, and b-adrenergic receptors in bone vascular smooth muscle and have confirmed that bone endothelium may secrete relaxing factors that affect vascular smooth muscle tone. In addition, the presence of endothelin receptors have been confirmed in canine bone blood vessels. Using an ex vivo perfused bone model, the effect of ischemic storage on both endothelial eccrine mechanisms and smooth muscle adrenergic receptor sensitivity has been clarified. Moreover, the most effective preservation technique to maintain the integrity of endothelial eccrine function has been studied. The major emphasis and specific aims of this proposal are to characterize the role of endothelins in bone blood vessels under ischemic and non-ischemic conditions and to confirm with in vivo methodology the results of prior studies herein described which employed an ex vivo perfused bone model. In order to appropriately evaluate the effect of endothelial and smooth muscle factors on bone blood flow control using a physiologically relevant in vivo model, studies of repetitive bone blood flow quantitation by serial microsphere injections are planned. This particular portion of the proposal will define the expected random error of the microsphere technique for repetitive blood flow studies in anesthetized dogs and suggest the ideal technique of bone blood flow quantitation using microspheres in in situ bones, fresh revascularized allograft bones, and stored revascularized allograft bones. The results of the investigations proposed for this funding period are expected to characterize the relative importance of both endothelial smooth muscle relaxing and constricting factors under non-ischemic and ischemic conditions and to clarify in vivo the effect of ischemic storage for 24 hours under ideal conditions on the bone vascular bed. Taken collectively with the results of studies already completed, the data derived from this project are expected to be of relevance for understanding the important factors of blood flow regulation in free vascularized bone autografts and possibly future applications of vascularized bone allografts. This information in turn may be useful to develop techniques to maximize bone tissue bed perfusion following microsurgical revascularization, and hence promote intraosseous cell survival, bone cell repopulation and ultimate bone healing. Much of this information may also be germane to a more comprehensive understanding of bone blood flow autoregulatory control under physiologic conditions.